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Cigarette smoking is a risk factor in the incidence and/or progression of periodontal
disease[1,2]. Numerous studies have examined the effects of whole tobacco smoke as well as individual components on periodontal tissues, such as epithelium
and connective tissue and on components of the inflammatory and immune response. A variety of studies have been carried
out to assess the changes in levels of inflammatory and host response substances caused by nicotine, a major component of
the particulate phase of all cigarette smoke. Previous studies have shown that nicotine is toxic to fibroblasts derived from
periodontium through inhibiting cell viability, attachment, proliferation, and matrix protein
synthesis[3-6]. Our recent studies have shown that nicotine can induce
c-fos[7], cyclooxygenase-2
(COX-2)[8], and heme
oxygenase-1[9] expression in fibroblasts
derived from perio-dontium. However, the mechanism behind the nicotine-induced expression of
c-fos, COX-2 or other signal proteins still remains to be elucidated.
COX is the rate-limiting enzyme that catalyzes the oxygenation of arachidonic acid to prostaglandin endoperoxi-dases
which are converted enzymatically into prostaglandin and thromboxane A2. Two distinct isoforms of COX have been
identified[10]. COX-1 is constitutively
expressed at a low level in most tissues but in contrast,
COX-2, the product of a related inducible gene, is absent from most tissues but is expressed in response to proliferative and inflammatory
stimuli[11,12].
Recently, we showed that nicotine could induce COX-2 mRNA and protein expression in human gingival fibroblasts
(HGF)[8]. As far as we know, little is known about whether chemical interactions can modulate nicotine-induced COX-2
expression. Antioxidants are substances that, when existing at low concentrations compared with those of the oxidizable
substrate, significantly delay or prevent oxidation of that
substrate[13]. To determine whether oxidative stress is important in
the induction of COX-2 expression by nicotine, we pretreated cells with the glutathione (GSH) precursor,
2-oxothiazolidine-4-carboxylic acid (OTZ), to boost thiol levels, or buthionine sulfoximine (BSO) to deplete GSH. In addition, oxidative stress
was found to activate extracellular signal-regulated protein kinase (ERK) signaling, which in turn has been implicated in the
response to inflammation[14]. Furthermore, ERK inhibitor PD98059 was added to investigate the possible regulation
mechanisms of nicotine-induced COX-2 expression. In the present study, we show that nicotine-induced COX-2 expression is
augmented by oxidative stress and mediated by ERK signaling.
Materials and methods
Reagents Nicotine, BSO (a cellular GSH synthesis inhibitor), and OTZ (a precursor of cysteine) were purchased from
Sigma (St Louis, MO, USA). PD98059 (an ERK inhibitor) was obtained from Promega (Madison, WI, USA). Mouse
anti-human COX-2 monoclonal antibody was obtained from Transduction Laboratories (Lexington, KY, USA). Anti-ERK and
anti-phospho-ERK were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Tissue culture reagents were
purchased from Gibco Laboratories (Grand Island, NY, USA). The final concentrations of nicotine, OTZ, BSO, and PD98059 used
in this study were 5 mmol/L, 5 mmol/L, 50 µmol/L, and 10 µmol/L, respectively.
Cell culture The human biopsy materials used in this study were obtained according to the guidelines of the Oral
Medicine Center and the Joint Research and Ethics Committee of the Chung Shan Medical University Hospital. Normal
gingival tissue samples were obtained from 3 healthy individuals undergoing routine surgical crown lengthening, who had
little if any evidence of inflammation, and who were receiving no systemic medication. HGF were cultured by using an explant
technique as described previously[9]. Cells were grown in Dulbecco¡¯s modified Eagle¡¯s medium (DMEM) supplemented with
10% fetal calf serum (FCS) and antibiotics (100
U/mL of penicillin, 100 µg/mL of streptomycin and
0.25 µg/mL of fungizone). Cultures were maintained at 37
¡ãC in a humidified atmosphere of 5% CO2
and 95% air. Confluent cells were detached with 0.25% trypsin and 0.05% EDTA for 5 min, and aliquots of separated cells were subcultured. Cell
cultures between the third and eighth passages were used.
Lactate dehydrogenase assessment Cellular toxicity was measured by the release of the cytoplasmic enzyme lactate
dehydrogenase (LDH). Briefly, cells were seeded at an initial density of
5¡Á105 in 60-mm culture dishes and allowed to attach
for 24 h. The cells were then incubated with nicotine at concentrations of
0-20 mmol/L. Cells were detached with
0.25% trypsin and 0.05% ethylenediamine tetraacetic acid (EDTA) for 5 min. To measure LDH, 500 µL of the cell suspension
was pelleted 24 h later. The LDH released into the supernatant was measured on the basis of reduced absorbance at 340 nm
due to NADH consumption during the reaction of pyruvate to lactate catalyzed by LDH. The percentage of LDH leakage in
response to nicotine as compared with untreated cells was calculated by the formula:
Chemical treatment Cells arrested in
G0 by serum deprivation (0.5% FCS; 48 h) were used in the
experiments[7,8]. Prior to treatment, the cells were washed with serum-free DMEM and immediately thereafter exposed to 5 mmol/L nicotine for the
indicated incubation times. Cells were pre-exposed to OTZ or BSO for 24 h before the addition of nicotine. This protocol was
necessary to allow changes in GSH levels to occur prior to exposure to nicotine. For COX-2 expression, cell lysates were
collected at 4 h. For ERK and phosphor ERK expression, cell lysates were collected at 0, 0.5, 1, 2, 4, and 8 h.
Western blotting For Western blot analysis, cell lysates were collected as described
previously[8,12]. Briefly, cells were solubilized with sodium dodecylsulfate (SDS) solubilization buffer [5 mmol/L EDTA, 1 mmol/L
MgCl2, 50 mmol/L Tris-HCl(pH 7.5) and 0.5% Trition X-100, 2 mmol/L phenylmethylsulfonyl fluoride, and 1 mmol/L
N-ethylmaleimide] for 30 min on ice. Then, cell lysates were centrifuged at 12
000¡Ág at 4 ¡ãC and the protein concentrations were determined with Bradford reagent using
bovine serum albumin (BSA) as standards. Equivalent amounts of total protein per sample of cell extracts were run on a 10%
sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and immediately transferred to nitrocellulose
membranes. The membranes were blocked with phosphate-buffered saline (PBS) containing 3% BSA for 2 h, rinsed, and then
incubated with primary antibodies anti-COX-2 (1:1000) or anti-ERK (1:1000) or antiphospho-ERK (1:1000) in PBS containing
0.05% Tween 20 for 2 h. After 3 washes with Tween 20 for 10 min, the membranes were incubated for 1 h with biotinylated
secondary antibody diluted at 1:1000 in the same buffer, washed again as described earlier and treated with 1:1000
streptavidin-peroxidase solution for 30 min. After a series of washing steps, the reactions were developed using Diaminobenzidine (DAB,
Zymed, South San Francisco, CA, USA). The intensities of the obtained bands were determined by a densitometer (AlphaImager
2000; Alpha Innotech, San Leandro, CA, USA). Each densitometric value was expressed as
mean¡ÀSD.
Statistical analysis Three replicates of each concentration were performed in each test. All assays were repeated 3 times
to ensure reproducibility. Statistical analysis was carried out by one-way analysis of variance (ANOVA). Tests of differences
of the treatments were analyzed by Duncan¡¯s test and a value of
P<0.05 was considered statistically significant.
Results
Leakage of LDH in HGF after exposure to nicotine for 24 h
Nicotine was found to elevate LDH leakage in a
dose-dependent manner (P<0.05). Nicotine was cytotoxic to HGF at concentrations of 5 mmol/L and above according to LDH assay
(Figure 1). The amount of LDH leakage was approximately 32%, 45%, 68%, and 96% at concentrations of 5, 10, 15, and 20
mmol/L, respectively (Figure 1).
COX-2 protein expression in HGF induced by 5 mmol/L
nicotine Nicotine upregulated COX-2 protein expression in
HGF. In addition, pretreatment with OTZ decreased the nicotine-induced COX-2 protein level by approximately 60%
(P<0.05). However, BSO enhanced the nicotine-induced COX-2 protein level by up to 3-fold
(P<0.05; Figure 2).
Effect of nicotine on ERK activation PD98059, a specific inhibitor of ERK, was used to block the activation of COX-2
expression. Treatment of HGF with PD98059 decreased the nicotine-induced COX-2 protein expression (Figure 3).
Nicotine induced ERK phosphorylation in a time-dependent manner
(P<0.05, Figure 4), with maximal activity appearing 2
h after initiating exposure to nicotine. Amounts of ERK protein were unaffected by nicotine during the same time interval. The
quantitative measurement of p-ERK protein was made by using the AlphaImager 2000 densitometer (Figure 5). The levels of
the p-ERK increased approximately 1.9-,
7.1-, 9.4-, 3.7- and 3.1-fold after exposure to nicotine for 0.5, 1, 2, 4, and 8 h, respectively. However, cells resting in 0.5% FCS
did not express detectable levels of p-ERK.
Discussion
Some of the components of tobacco products, for example nicotine, may contribute to the development of periodontal
disease via direct effects on cell viability, host maintenance, and the cellular defense system. Normal fibroblast function is
critical for the maintenance of periodontal tissues and for optimal wound healing responses. HGF were chosen for the present
study due to their ready availability and particular culturing characters. Periodontal ligament fibroblasts would stimulate
periodontal tissues better, but these cells are known to be similar to
HGF[15]. In addition, Aukhil et
al[16] have reported that only a very limited area of
200-400 µm of the periodontal ligament apical to the surgical wound actually participates as a
source of regenerative cells. Thus, the effects of nicotine on HGF may have clinical significance.
In the present study, the cytotoxicity of nicotine was evaluated using an LDH leakage assay in HGFs. It was found that
nicotine was toxic to HGF. Although the experimental conditions in the present study differed from those used in other
studies, our results were in agreement with previous studies in that we found that nicotine was cytotoxic to human fibroblasts
derived from periodontium[3-6]. It has been suggested that factors that inhibit the functions of HGF would also impair tissue
repair and regeneration. Tobacco users may be more susceptible to destruction of the periodontium and less responsive to
regeneration procedures during periodontal therapy.
COX-2 is an inducible enzyme that is believed to be responsible for prostaglandin synthesis at sites of inflamma-tion.
Data from our in vitro experiments confirmed our previous report that nicotine was capable of stimulating COX-2 protein
expression in HGF[10]. Our results are also in agreement with those of Yan
et al[17], who reported that
benzo[a]pyrene, a polycyclic aromatic hydrocarbon found in tobacco smoke, induced the transcription of COX-2 in vascular smooth muscle
cells, and of Anto et al[18], who demonstrated that cigarette smoke condensate induced COX-2 expression in human
epithelial, lymophoid, and myeloid cells in
vitro. Thus, one of the pathogenetic mechanisms of chronic periodontal
inflammation may be the synthesis of COX-2 by resident cells in cigarette smokers.
The sulfphhydryl group containing tripeptide constitutes a first-defense intracellular
antioxidant[19]. GSH plays a role in cellular protection from damage produced by free radicals and electrophiles. It is well-known BSO, a specific inhibitor of
g-glutamyl cysteine synthetase, inhibits GSH synthesis, is relatively non-toxic, and is quite efficient in decreasing intracellular
GSH levels. OTZ, a precursor of cysteine, meta
bolically promotes GSH synthesis, increasing intracellular GSH levels by as much as 2 to 3 times the control
level[20].
In the present study, we found that the extent of COX-2 induction by nicotine in HGF depended upon the antioxidant
potential of the HGF, being strongly enhanced when GSH was depleted by pretreatment with BSO, an inhibitor of GSH
synthesis, and strongly diminished by treatment with OTZ, a GSH synthesizer. These data suggest that the oxidant effects
of nicotine mediate the induction of COX-2 in HGF, a finding consistent with previous reports that oxidative stress can
induce COX-2[17,21]. These results suggest that thiol pools may act as an intracellular buffer against nicotine-induced
COX-2 expression.
COX-2 expression has been linked with the activation of the ERK signaling transduction
pathway[22,23]. To further investigate the mechanism of nicotine-induced signaling proteins, Western blot analysis of the phosphorylated proteins
was performed. Phosphorylation of ERK was increased, whereas no increase was observed in ERK. COX-2 induction via
preferential activation of ERK has been associated with resistance to oxidative stress in smooth muscle
cells[17]. Recently, similar results were found by Huang
et al[24] who reported that nicotine could directly induce p-ERK expression in human
osteosarcoma cells. Taken together, these data provide the first evidence that activation of ERK pathway is directly involved
in the nicotine-induced COX-2 expression.
Data from the present study indicate that the effect of nicotine in HGF is through activation of the COX-2 pathway.
COX-2 expression is critically dependent on cellular thiol levels allowing nicotine to interfere with specific target molecules
resulting in the expression of COX-2. COX-2 expression is directly involved in the phosphorylation of ERK. Thus,
nicotine-induced COX-2 expression is augmented by oxidative stress and mediated by ERK signaling. Understanding the cellular
processes involved and the molecular mechanisms responsible for COX-2 induction by nicotine may aid in the development
of more effective treatments for cigarette smoking-associated periodontal disease. Factors that induce glutathione
synthesis or ERK inhibitors suppress COX-2 expression and may therefore be clinically useful agents, in combination with standard
treatment modalities, in the treatment of COX-2-mediated cigarette smoking-associated periodontal destruction.
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